IgE antagonists

ABSTRACT

The present invention relates to methods for modulating interaction between IgE and FcεR1. The present invention also relates to compounds that modulate IgE binding to FcεR1.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Phase of PCT/US95/08401, filedJun. 30, 1995, and a Continuation In Part of U.S. Ser. No. 08/271,943,filed Jul. 8, 1994, abandoned.

FIELD OF THE INVENTION

The present invention relates to biologically active compounds which aresimilar to regions of the high affinity Fc receptor for immunoglobulinE.

BACKGROUND OF THE INVENTION

Clinical bone marrow transplantation is an important therapeutictreatment for several diseases including high risk leukemia, aplasticanemia, and severe combined immunodeficiency. In addition, there is awide range of metabolic and genetic disorders that can potentially becorrected by this approach. However, the usefulness of marrowtransplantation is currently limited by several important risk factors,the principal one being graft-versus-host disease (GVHD), an often timeslethal complication which occurs in a high proportion of transplants.

The risk of GVHD can be reduced by HLA matching of the marrow donor andrecipient, with a matched sibling being the primary choice. Yet, lessthan 30% of the patients in North America have an HLA-matched sibling,and therefore must seek suitable unrelated HLA-matched donors from theNational Marrow Donor Program. The probability of finding an unrelatedHLA-matched donor is currently on the order of 30-40% and depend on thetotal number of donors registered. In both related and unrelatedHLA-matched transplant situations, the risk of GVHD is still quite highdue to disparity of non-HLA multiple minor histocompatibility (H)antigens. GVHD is somewhat higher in unrelated cases, as this increasesthe probability of differences at these loci.

Mature donor T cells contaminating the marrow inoculum are responsiblefor GVHD. Several studies have shown that depletion of these T cellssignificantly diminishes the incidence of disease. However, theelimination of donor T cells has also resulted in a greater incidence ofleukemic relapse. It seems important to provide at least some level of Tcell immunocompetency in these completely immunocompromised patients tonot only combat residual leukemia cells but also to counteropportunistic infections. In this respect, the same GVHD-reactive donorT cells may be important for targeting leukemia cells expressing thesame host allogeneic histocompatibility antigens. Therapeutic approachesthat could ameliorate the pathogenic tissue destruction accompanyingGVHD, particularly in the gut and skin, but that would allow forcontinued anti-leukemia activity would greatly benefit marrow transplantpatients.

Immediate allergic responses, also referred to as type 1hypersensitivity reactions, are mediated through the interaction ofimmunoglobulin E (IgE) with the α-chain of its high affinity Fcreceptor, FcεR1. The binding of a multivalent allergen to anIgE-FcεR1complex initiates a cross-linking of the receptor andconsequent cellular activation. These high affinity receptors are foundprimarily on mast cells and basophils. When activated by an allergen,these cells respond by releasing histamine, eicosanoids and cytokines.

In addition to its high affinity Fc receptor, IgE binds to a lowaffinity receptor, the FcεR2, which is found on B cells, T cells,macrophages, NK cells, eosinophils, platelets, follicular dendriticcells as well as several other cell types. Activation of the FcεR2 hasbeen implicated in IgE-dependent cell cytotoxicity (ADCC) as well as inallergic inflammation.

Many different fine mapping studies have been conducted on the bindingof the IgE to its Fc receptors. The consensus mapping data is confirmedby a recent study by Nissim et al. (1993) J. Immunol. 150: 1365-1374,which is incorporated herein by reference, showing that the bindingactivity for both the high and low affinity receptors resides within theCε3 (the third constant domain) of the IgE. Data show that the speciesspecific binding of IgE to the FcεR1 is contained within the first 16amino acids of the CE3, whereas no species specific binding is observedin the same region with respect to binding the FcεR2.

There is a need for compounds and methods which can inhibit IgE-FcεR1interaction. IgE-FcεR1 interaction is associated with immediate allergicreactions. There is a need for compounds and methods which can inhibitmast cell and basophil activity that results from IgE-FcεR1 interaction.Mast cell and basophil activity linked to IgE-FcεR1 interaction isassociated, for example with reactions to allergens and GVHD.Identification of compounds that inhibit the IgE-FcεR1 interaction canbe used in methods of treating allergies and GVHD.

SUMMARY OF THE INVENTION

The present invention relates to peptides that modulate IgE binding toFcεR1 and that consist of 4-50 amino acids and comprise at least afragment of SEQ ID NO:3 of at least 4 amino acids.

The present invention relates to pharmaceutical compositions comprisinga peptide that modulate IgE binding to FcεR1and that consist of 4-50amino acids and comprises at least a fragment of SEQ ID NO:3 of at least4 amino acids and a pharmaceutically acceptable carrier or diluent.

The present invention relates to peptides that modulate IgE binding toFcεR1 and that consist of 6-50 amino acids and comprise SEQ ID NO:5.

The present invention relates to pharmaceutical compositions comprisinga peptide that modulate IgE binding to FcεR1and that consist of 6-50amino acids and comprise SEQ ID NO:5 and a pharmaceutically acceptablecarrier or diluent.

The present invention relates to a method of inhibiting IgE binding toFcεR1on cells comprising the steps of contacting cells that contain theFcεR1 with a peptide that inhibits IgE binding to FcεR1 and thatconsists of 4-50 amino acids and comprises at least a fragment of SEQ IDNO:3 of at least 4 amino acids in an amount sufficient to inhibit IgEbinding to FcεR1.

The present invention relates to a method of inhibiting IgE binding toFcεR1 on cells comprising the steps of contacting cells that contain theFcεR1 with a peptide that inhibits IgE binding to FcεR1 and thatconsists of 6-50 amino acids and comprises SEQ ID NO:5 in an amountsufficient to inhibit IgE binding to FcεR1.

The present invention relates to a method of therapeutically orprophylactically treating an individual suffering from or susceptible toan immediate allergic response comprising administering to such anindividual an effective amount of a peptide that inhibits IgE binding toFcεR1 and that consists of 4-50 amino acids and comprises at least afragment of SEQ ID NO:3 of at least 4 amino acids in an amountsufficient to inhibit IgE binding to FcεR1.

The present invention relates to a method of therapeutically orprophylactically treating an individual suffering from or susceptible toan immediate allergic response comprising administering to such anindividual an effective amount of a peptide that inhibits IgE binding toFcεR1 and that consists of 6-50 amino acids and comprises SEQ ID NO:5 inan amount sufficient to inhibit IgE binding to FcεR1.

The present invention relates to a peptide dimer that modulate IgEbinding to FcεR1 comprising a first monomer which consists of between 4and 50 amino acids and comprises at least a fragment of SEQ ID NO:3 ofat least 4 amino acids, and a second monomer which consists of between 6and 50 amino acids and comprises SEQ ID NO:5.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows data from in vitro experiments testing the effect ofvarious concentrations of D amino acid peptide #1, which is described inExample 3, on the level of inhibition of IgE-IgE receptor.

FIG. 2 shows data from in vivo experiments testing the effect of D aminoacid peptide #1, which is described in Example 3, on survival of mice ina GVHD model.

FIG. 3 shows data from inhibition assays using a biosensor in which theassociation of IgE-Fc at a concentration of 5nM was monitored for 6minutes in the presence at various concentrations and absence of abiotinylated derivative of the D amino acid peptide #1, which isdescribed in Example 3.

FIG. 4 shows the in vitro inhibition of binding of IgE Fc to Fcε bybiosensor assay.

FIG. 5 shows in vivo data that demonstrates inhibition of GVDH bycompounds of the invention.

FIG. 6 shows in vivo data that demonstrates inhibition ofantigen-induced anaphylaxis by compounds of the invention.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides conformationally restrained analogs whichare modelled from the FcεR1 α-chain surface. This application is relatedto U.S. patent application Ser. No. 08/271,943 which is incorporated inits entirety herein by reference. Using commercially available molecularmodeling software, a structural model of the α-chain of the IgE highaffinity receptor was created using the known three-dimensionalcoordinates of related proteins (See Beavil, A. J. et al. 1993 Biochem.Soc. Trans. 21: 968-972, which is incorporated herein by reference). CD2was chosen as the principal template because its three dimensionalstructure was known and it shares significant sequence homology with theIgE- receptor protein.

The compounds of the invention are capable of inhibiting IgE-IgEreceptor binding linked to immediate allergic responses. The compoundsof the invention intervene in the IgE-IgE receptor signal pathway and,thereby, creating therapeutic reagents for the treatment ofIgE-dependent disease states including allergic rhinitis, foodallergies, atropic dermatitis and allergic asthma.

The amino acid sequence of FcεR1 is shown in SEQ ID NO:1. The amino acidsequence of CD2 is shown in SEQ ID NO:2. The known three dimensionalstructure of CD2 was used as a template. The FcεR1 amino acid sequenceshown in SEQ ID NO:1 was aligned to the three dimensional CD2 template.Alignment of the sequences allowed for a prediction of the threedimensional structure of FcεR1. The FcεR1 IgE-binding region in theα-chain sequence was identified using the predicted three dimensionalstructure. The modeled structure of the α-chain of the high affinity IgEreceptor, in turn, was used as a template in selecting and designingpeptides, particularly conformationally restrained analogs of thereceptor surface.

The location of a principal IgE-binding region in the α-chain of FcεR1occurs between amino acids 140 and 160 (134-154 of SEQ ID NO:1), moreparticularly between amino acids 144-154 (139-149 of SEQ ID NO:1) of theFcεR1 protein sequence. The amino acid sequence 144-154 (139-149 of SEQID NO:1) is shown as SEQ ID NO:3.

According to some embodiments, the peptides of the invention thatinhibit IgE from binding to FcεR1 consist of 4-50 amino acids andcomprise at least a fragment of SEQ ID NO:3 of at least 4 amino acids.

In some preferred embodiments, the peptides of the invention consist of4-25 amino acids including at least a fragment of SEQ ID NO:3 of atleast 4 amino acids. In some embodiments, the peptide of the inventionconsist of 6-20 amino acids including at least a fragment of SEQ ID NO:3of at least 4 amino acids. In some embodiments, the peptide of theinvention consist of 8-15 amino acids including at least a fragment ofSEQ ID NO:3 of at least 4 amino acids. In some embodiments, the peptidesof the invention consist of 10-12 amino acid residues including at leasta fragment of SEQ ID NO:3 of at least 4 amino acids.

As used herein, the term "fragment of SEQ ID NO:3" is meant to refer topeptides which comprise an amino acid sequence identical to a portion ofSEQ ID NO:3 having at least 4. In some embodiments, peptides thatcomprise a fragment of SEQ ID NO:3 include fragments of SEQ ID NO:3having 4, 5, 6, 7, 8, 9 or 10. Accordingly, a fragment of SEQ ID NO:3 ofat least 4 amino acids may be have amino acids 1-4, 2-5, 3-6, 4-7, 5-8,6-9, 7-10, 8-11, 1-5, 2-6, 3-7, 4-8, 5-9, 6-10, 7-11, 1-6, 2-7, 3-8,4-9, 5-10, 6-11, 1-7, 2-8, 3-9, 4-10, 5-11, 1-8, 2-9, 3-10, 4-11, 1-9,2-10, 3-11, 1-10 or 2-11 of SEQ ID NO:3. Peptides may comprise SEQ IDNO:3. Thus, a peptide of the invention that comprises at least afragment of SEQ ID NO:3 of at least 4 amino acids may comprise SEQ IDNO:3 or a fragment thereof such as one that has amino acids 1-4, 2-5,3-6, 4-7, 5-8, 6-9, 7-10, 8-11, 1-5, 2-6, 3-7, 4-8, 5-9, 6-10, 7-11,1-6, 2-7, 3-8, 4-9, 5-10, 6-11, 1-7, 2-8, 3-9, 4-10, 5-11, 1-8, 2-9,3-10, 4-11, 1-9, 2-10, 3-11, 1-10 and 2-11 of SEQ ID NO:3. In somepreferred embodiments, the peptides of the invention comprise SEQ IDNO:3. In some preferred embodiments, the peptides comprise a fragment ofSEQ ID NO:3 that consists of 4-10 amino acids. In some embodiments, thepeptides comprise a fragment of SEQ ID NO:3 that consists of 6-10 aminoacids. In some embodiments, the peptides comprise a fragment of SEQ IDNO:3 that consists of 8-10 amino acids.

In addition to SEQ ID NO:3 or fragments thereof, peptides of theinvention may comprise at least a fragment of SEQ ID NO:3 of at least 4amino acids and further comprise additional amino acids. The additionalamino acids may be FcεR1 amino acids, non-FcεR1 amino acids or both.FcεR1 amino acids include amino acids 140-143 (amino acids 134-137 ofSEQ ID NO:1) or fragments thereof, amino acids 155-160 (amino acids149-154 of SEQ ID NO:1) or fragments thereof. Additional FcεR1 aminoacids include adjacent amino acid sequences. Moreover, peptides maycomprise other FcεR1 amino acid sequences such as those which defineother IgE-FcεR1 binding regions. Non-FcεR1 amino acids include aminoacids used to conformationally restrict the peptide.

In some preferred embodiments, peptides are conformationally constrainedand consist of amino and carboxy terminal cysteines flanking SEQ ID NO:3or a fragment thereof selected from the group consisting of amino acids1-4, 2-5, 3-6, 4-7, 5-8, 6-9, 7-10, 8-11, 1-5, 2-6, 3-7, 4-8, 5-9, 6-10,7-11, 1-6, 2-7, 3-8, 4-9, 5-10, 6-11, 1-7, 2-8, 3-9, 4-10, 5-11, 1-8,2-9, 3-10, 4-11, 1-9, 2-10, 3-11, 1-10 or 2-11 of SEQ ID NO:3. One ormore residues of such peptides may be a D amino acid. Peptides may bebiotinylated. The amino acid sequences described herein may beconstructed to proceed in sequence from the amino terminus to carboxyterminus or from the carboxy terminus to the amino terminus. When thesequence proceeds from the amino terminus to carboxy terminus, it isusually constructed of L amino acids. In some embodiments, it isconstructed of all L amino acids except either an N terminal cysteine ora C terminal cysteine which is a D amino acid. When D amino acids areused to construct the compound, the sequence is usually reversed fromthe order it occurs as an L amino acid peptide. It therefore proceedsfrom the carboxy terminus to the amino terminus. In some embodiments, itis constructed of all D amino acids except either an N terminal cysteineor a C terminal cysteine which is an L amino acid.

The location of another IgE-binding region of FCεR1 occurs between aminoacids 168-186 of SEQ ID NO:1, more particularly between amino acids174-179 of SEQ ID NO:1. The amino acid sequence 168-186 of SEQ ID NO:1is shown as SEQ ID NO:4. The amino acid sequence 174-179 of SEQ ID NO:1is shown as SEQ ID NO:5.

In some embodiments, the peptides of the invention that inhibit IgE frombinding to FcεR1 consist of 6-50 amino acids and comprises SEQ ID NO:5.

In some preferred embodiments, the peptides of the invention consist of6-25 amino acids including SEQ ID NO:5. In some embodiments, thepeptides consist of 6-20 amino acids including SEQ ID NO:5. In someembodiments, the peptides consist of 8-15 amino acids including SEQ IDNO:5. In some embodiments, the peptides consist of 10-12 amino acidsincluding SEQ ID NO:5.

In some embodiments, peptides of the invention which comprise SEQ IDNO:5 may comprise SEQ ID NO:4 or a fragment of SEQ ID NO:4 of at least 6amino acids including SEQ ID NO:5. As used herein, the term "fragment ofSEQ ID NO:4 of at least 6 amino acids including SEQ ID NO:5" is meant torefer to peptides which comprise an amino acid sequence identical to aportion of SEQ ID NO:4 having at least 6 including amino acids 7-12 ofSEQ ID NO:4. Amino acids 7-12 of SEQ ID NO:4 are SEQ ID NO:5. In someembodiments, peptides that comprise a fragment of SEQ ID NO:4 having atleast 6 amino acids including SEQ ID NO:5 include fragments of SEQ IDNO:4 having 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 aminoacids. Accordingly, a fragment of SEQ ID NO:4 of at least 6 amino acidsincluding SEQ ID NO:5 may be have amino acids 7-12, 7-13, 7-14, 7-15,7-16, 7-17, 7-18, 7-19, 6-12, 6-13, 6-14, 6-15, 6-16, 6-17, 6-18, 6-19,5-12, 5-13, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 4-12, 4-13, 4-14, 4-15,4-16, 4-17, 4-18, 4-19, 3-12, 3-13, 3-14, 3-15, 3-16, 3-17, 3-18, 3-19,2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 1-12, 1-13, 1-14, 1-15,1-16, 1-17, 1-18 or 1-19 of SEQ ID NO:4. Peptides may comprise SEQ IDNO:4. Thus, a peptide of the invention that comprises at least afragment of SEQ ID NO:4 of at least 6 amino acids including SEQ ID NO:5may comprise SEQ ID NO:4 or a fragment thereof such as one that hasamino acids 7-12, 7-13, 7-14, 7-15, 7-16, 7-17, 7-18, 7-19, 6-12, 6-13,6-14, 6-15, 6-16, 6-17, 6-18, 6-19, 5-12, 5-13, 5-14, 5-15, 5-16, 5-17,5-18, 5-19, 4-12, 4-13, 4-14, 4-15, 4-16, 4-17, 4-18, 4-19, 3-12, 3-13,3-14, 3-15, 3-16, 3-17, 3-18, 3-19, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17,2-18, 2-19, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18 and 1-19 of SEQ IDNO:4. In some preferred embodiments, the peptides comprise a fragment ofSEQ ID NO:4 that consists of 6-10 amino acids including SEQ ID NO:5. Insome embodiments, the peptides comprise a fragment of SEQ ID NO:4 thatconsists of 6-10 amino acids including SEQ ID NO:5. In some embodiments,the peptides comprise a fragment of SEQ ID NO:4 that consists of 8-10amino acids including SEQ ID NO:5.

In addition to SEQ ID NO:5 or fragments of SEQ ID NO:4 that comprise SEQID NO:5, according to some embodiments, peptides of the invention maycomprise additional amino acids. The additional amino acids may be FcεR1amino acids, non-FcεR1 amino acids or both. Additional FcεR1 amino acidsinclude adjacent amino acid sequences such as amino acids 150-167 and187-200 of SEQ ID NO:1 or fragments thereof. Moreover, peptides maycomprise other FcεR1 amino acid sequences such as those which defineother IgE-FcεR1 binding regions. Non-FcεR1amino acids include aminoacids used to conformationally restrict the peptide.

In some preferred embodiments, peptides are conformationally constrainedand consist of amino and carboxy terminal cysteines flanking SEQ ID NO:4or a fragment thereof selected from the group consisting of amino acids7-12, 7-13, 7-14, 7-15, 7-16, 7-17, 7-18, 7-19, 6-12, 6-13, 6-14, 6-15,6-16, 6-17, 6-18, 6-19, 5-12, 5-13, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19,4-12, 4-13, 4-14, 4-15, 4-16, 4-17, 4-18, 4-19, 3-12, 3-13, 3-14, 3-15,3-16, 3-17, 3-18, 3-19, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19,1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18 or 1-19 of SEQ ID NO:4. One ormore residues of such peptides may be a D amino acid. Peptides may bebiotinylated. The amino acid sequences described herein may beconstructed to proceed in sequence from the amino terminus to carboxyterminus or from the carboxy terminus to the amino terminus. When thesequence proceeds from the amino terminus to carboxy terminus, it isusually constructed of L amino acids. In some embodiments, it isconstructed of all L amino acids except either an N terminal cysteine ora C terminal cysteine which is a D amino acid. When D amino acids areused to construct the compound, the sequence is usually reversed fromthe order it occurs as an L amino acid peptide. It therefore proceedsfrom the carboxy terminus to the amino terminus. In some embodiments, itis constructed of all D amino acids except either an N terminal cysteineor a C terminal cysteine which is an L amino acid.

According to some embodiments of the invention, peptide dimers areprovided which comprise two different monomeric subunits, one of whichis a peptide of the invention that comprises at least a fragment of SEQID NO:3 of at least 4 amino acids and the other of which is a peptide ofthe invention which comprises SEQ ID NO:5. Dimeric peptides of theinvention inhibit IgE from binding to FcεR1 and comprise one monomerthat consists of 4-50 amino acids including at least a fragment of SEQID NO:3 of at least 4 amino acids, and one monomer that consists of 6-50amino acids including SEQ ID NO:5.

Dimers according to the invention comprise two monomeric subunits linkedto each other by non-peptide bonds. In some preferred dimers, each ofthe monomeric subunits has a cysteine residue at at least one terminusand the dimer is formed by disulfide bonds which form between a terminalcysteines of each monomer. In some preferred dimers, the monomericsubunits are linked by other covalent bonds between terminal residues.Methods of linking monomers to form dimers are well known to thosehaving ordinary skill in the art.

In some embodiments, the amino terminal of the monomer that comprises atleast a fragment of SEQ ID NO:3 is linked to the amino terminal of themonomer that comprises SEQ ID NO:5. In some embodiments, the aminoterminal of the monomer that comprises at least a fragment of SEQ IDNO:3 is linked to the carboxy terminal of the monomer that comprises SEQID NO:5. In some embodiments, the carboxy terminal of the monomer thatcomprises at least a fragment of SEQ ID NO:3 is linked to the aminoterminal of the monomer that comprises SEQ ID NO:5. In some embodiments,the carboxy terminal of the monomer that comprises at least a fragmentof SEQ ID NO:3 is linked to the carboxy terminal of the monomer thatcomprises SEQ ID NO:5.

In preferred embodiments of the invention which relate to dimers, themonomer that comprises at least a fragment of SEQ ID NO:3 of at least 4amino acids consists of 4-25 amino acids. In some embodiments, themonomer that comprises at least a fragment of SEQ ID NO:3 consists of6-20 amino acids. In some embodiments, the monomer that comprises atleast a fragment of SEQ ID NO:3 consists of 8-15 amino acids. In someembodiments, the monomer that comprises at least a fragment of SEQ IDNO:3 consists of 10-12 amino acid residues.

In preferred embodiments of the invention which relate to dimers, themonomer that comprises at least a fragment of SEQ ID NO:3 of at least 4amino acids comprises SEQ ID NO:3 or a fragment thereof such as one thathas amino acids 1-4, 2-5, 3-6, 4-7, 5-8, 6-9, 7-10, 8-11, 1-5, 2-6, 3-7,4-8, 5-9, 6-10, 7-11, 1-6, 2-7, 3-8, 4-9, 5-10, 6-11, 1-7, 2-8, 3-9,4-10, 5-11, 1-8, 2-9, 3-10, 4-11, 1-9, 2-10, 3-11, 1-10 and 2-11 of SEQID NO:3.

In preferred embodiments of the invention which relate to dimers, themonomer that comprises SEQ ID NO:5 consists of 6-25 amino acids. In someembodiments, the monomer which comprises SEQ ID NO:5 consists of 6-20amino acids. In some embodiments, the monomer that comprises SEQ ID NO:5consists of 8-15 amino acids. In some embodiments, the monomer whichcomprises SEQ ID NO:5 consists of 10-12 amino acid residues.

In preferred embodiments of the invention which relate to dimers, themonomer that comprises SEQ ID NO:5 comprises SEQ ID NO:4 or a fragmentthereof such as one that has amino acids 7-12, 7-13, 7-14, 7-15, 7-16,7-17, 7-18, 7-19, 6-12, 6-13, 6-14, 6-15, 6-16, 6-17, 6-18, 6-19, 5-12,5-13, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 4-12, 4-13, 4-14, 4-15, 4-16,4-17, 4-18, 4-19, 3-12, 3-13, 3-14, 3-15, 3-16, 3-17, 3-18, 3-19, 2-12,2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 1-12, 1-13, 1-14, 1-15, 1-16,1-17, 1-18 and 1-19 of SEQ ID NO:4.

In preferred embodiments of the invention which relate to dimers, themonomer that comprises at least a fragment of SEQ ID NO:3 consists of4-25 amino acids and the monomer that comprises SEQ ID NO:5 consists of6-25 amino acids. In some embodiments, the monomer that comprises atleast a fragment of SEQ ID NO:3 of at least 4 amino acids consists of6-20 amino acids and the monomer which comprises SEQ ID NO:5 consists of6-20 amino acids. In some embodiments, the monomer that comprises atleast a fragment of SEQ ID NO:3 of at least 4 amino acids consists of8-15 amino acids and the monomer that comprises SEQ ID NO:5 consists of8-15 amino acids. In some embodiments, the monomer that comprises atleast a fragment of SEQ ID NO:3 of at least 4 amino acids consists of10-12 amino acid residues and the monomer which comprises SEQ ID NO:5consists of 10-12 amino acid residues.

In some preferred embodiments of the invention which relate to dimers,the monomer that comprises at least a fragment of SEQ ID NO:3 of atleast 4 amino acids comprises SEQ ID NO:3 or a fragment thereof such asone that has amino acids 1-4, 2-5, 3-6, 4-7, 5-8, 6-9, 7-10, 8-11, 1-5,2-6, 3-7, 4-8, 5-9, 6-10, 7-11, 1-6, 2-7, 3-8, 4-9, 5-10, 6-11, 1-7,2-8, 3-9, 4-10, 5-11, 1-8, 2-9, 3-10, 4-11, 1-9, 2-10, 3-11, 1-10 and2-11 of SEQ ID NO:3 and the monomer that comprises SEQ ID NO:5 comprisesSEQ ID NO:4 or a fragment thereof such as one that has amino acids 7-12,7-13, 7-14, 7-15, 7-16, 7-17, 7-18, 7-19, 6-12, 6-13, 6-14, 6-15, 6-16,6-17, 6-18, 6-19, 5-12, 5-13, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 4-12,4-13, 4-14, 4-15, 4-16, 4-17, 4-18, 4-19, 3-12, 3-13, 3-14, 3-15, 3-16,3-17, 3-18, 3-19, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 1-12,1-13, 1-14, 1-15, 1-16, 1-17, 1-18 and 1-19 of SEQ ID NO:4.

According to some embodiments of the invention, peptides that inhibitIgE from binding to FcεR1 are provided which comprise at least afragment of SEQ ID NO:3 of at least 4 amino acids and which furthercomprises SEQ ID NO:5. More particularly, in some embodiments of theinvention, peptides comprise 10-100 amino acids including at least afragment of SEQ ID NO:3 of at least 4 amino acids and SEQ ID NO:5.

In some preferred embodiments of the invention, the peptides, whichinclude at least a fragment of SEQ ID NO:3 of at least 4 amino acids andSEQ ID NO:5, comprise 10-50 amino acids including at least a fragment ofSEQ ID NO:3 having 4-11 amino acids and at least a fragment of SEQ IDNO:4 having 6-19 amino acids including SEQ ID NO:5. In some embodiments,the peptides, which include at least a fragment of SEQ ID NO:3 of atleast 4 amino acids and SEQ ID NO:5, comprise 12-40 amino acidsincluding at least a fragment of SEQ ID NO:1 of 6-20 amino acidsincluding 6-11 amino acids from SEQ ID NO:3. In some embodiments, thepeptide, which include at least a fragment of SEQ ID NO:3 of at least 4amino acids and SEQ ID NO:5, comprise 14-30 amino acids including atleast a fragment of SEQ ID NO:1 of 8-15 amino acids including 8-11 aminoacids from SEQ ID NO:3. In some embodiments, the peptide, which includeat least a fragment of SEQ ID NO:3 of at least 4 amino acids and SEQ IDNO:5, comprises 16-25 amino acids including at least a fragment of SEQID NO:1 of 11-12 amino acids including SEQ ID NO:3. In some embodiments,the peptide, which include at least a fragment of SEQ ID NO:3 of atleast 4 amino acids and SEQ ID NO:5, comprises 12-40 amino acidsincluding SEQ ID NO:5. In some embodiments, the peptide, which includeat least a fragment of SEQ ID NO:3 of at least 4 amino acids and SEQ IDNO:5, comprises 14-30 amino acids including SEQ ID NO:5. In someembodiments, the peptide, which include at least a fragment of SEQ IDNO:3 of at least 4 amino acids and SEQ ID NO:5, comprises 16-25 aminoacids including SEQ ID NO:5. In some embodiments, the peptide, whichinclude at least a fragment of SEQ ID NO:3 of at least 4 amino acids andSEQ ID NO:5, comprises SEQ ID NO:3 or a fragment thereof such as onethat has amino acids 1-4, 2-5, 3-6, 4-7, 5-8, 6-9, 7-10, 8-11, 1-5, 2-6,3-7, 4-8, 5-9, 6-10, 7-11, 1-6, 2-7, 3-8, 4-9, 5-10, 6-11, 1-7, 2-8,3-9, 4-10, 5-11, 1-8, 2-9, 3-10, 4-11, 1-9, 2-10, 3-11, 1-10 and 2-11 ofSEQ ID NO:3 linked to SEQ ID NO:5 comprises SEQ ID NO:4 or a fragmentthereof such as one that has amino acids 7-12, 7-13, 7-14, 7-15, 7-16,7-17, 7-18, 7-19, 6-12, 6-13, 6-14, 6-15, 6-16, 6-17, 6-18, 6-19, 5-12,5-13, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 4-12, 4-13, 4-14, 4-15, 4-16,4-17, 4-18, 4-19, 3-12, 3-13, 3-14, 3-15, 3-16, 3-17, 3-18, 3-19, 2-12,2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 1-12, 1-13, 1-14, 1-15, 1-16,1-17, 1-18 and 1-19 of SEQ ID NO:4.

In some embodiments, the peptide which includes the sequence which isdefined as at least a fragment of SEQ ID NO:3 of at least 4 amino acidsat the amino terminal portion of the sequence relative to the positionof the portion of the sequence that comprises SEQ ID NO:5.

In some embodiments, the peptide which includes the sequence SEQ ID NO:5at the amino terminal portion of the sequence relative to the positionof the portion of the sequence that which is defined as the sequencethat comprises at least a fragment of SEQ ID NO:3 of at least 4 aminoacids.

Peptides of the invention inhibit IgE from binding to FcεR1. The abilityof a peptide to inhibit IgE from binding to FcεR1 may be determined byone having ordinary skill in the art by performing binding assays whichare in vitro and/or in vivo assays using readily available startingmaterials. For example, assays to determine whether a peptide hasbiological activity are described in Examples 1 and 2. Briefly, an invitro assay is described which allows one to determine whether or notIgE is inhibited from binding to FcεR1. An in vivo assay is describedwhich allows one to determine whether or not GVHD occurs in a bonemarrow transplant model. GVHD is linked to IgE dysregulation and to mastcell activity (Claman and Spiegelberg 1990 Clin. Imm. Immunopath. 56:46-53; and Murphy et al. 1994 J. Invest. Derma. 102: 451-461).Accordingly, inhibition of IgE-FcεR1 binding in vivo will prevent thelethal effects of GVHD in the in vivo assay. The ability of a compoundof the invention to inhibit IgE from binding to FcεR1 IgE refers itspossession of IgE-FcεR1binding inhibition activity in an IgE-FcεR1binding assay.

In some embodiments, peptides of the present invention comprise D aminoacids. As used herein, the term "D amino acid peptides" is meant torefer to peptides according to the present invention which comprise atleast one and in some embodiments a plurality of D amino acids. D aminoacid peptides inhibit IgE from binding to FcεR1, thus retaining thebiological activity of peptides having L amino acid sequences. The useof D amino acid peptides is desirable as they are less vulnerable todegradation and therefore have a longer half life. D amino acid peptidescomprising mostly all or consisting of D amino acids may comprise aminoacid sequences in the reverse order of SEQ ID NO:3 or fragments orderivatives thereof or SEQ ID NO:4 or fragments thereof.

In some embodiments, peptides of the present invention areconformationally restricted to present and maintain the properstructural conformation for biological activity. The compositions maycomprise additional amino acid residues required to achieve proper threedimensional conformation including residues which facilitatecircularization or desired folding. Accordingly, in preferredembodiments, cysteine residues are added to the terminal residues ofpeptides comprising SEQ ID NO:3 or fragments thereof or SEQ ID NO:4 offragments thereof including SEQ ID NO:5. Formation of disulfide bondsbetween the terminal cysteines results in a circularized, i.e. cyclicpeptides.

D amino acid residues may be provided to facilitate conformationalrestriction. For example, in order to facilitate disulfide bondformation and stability, a D amino acid cysteine may be provided at oneor both termini.

Peptides which fall within the structural definition provided herein maybe produced by those having ordinary skill in the art using routinetechniques and readily available starting materials without undueexperimentation. One having ordinary skill in the art can then test thepeptides to determine whether or not they inhibit IgE binding to FcεR1and are thus peptides of the invention.

It is also contemplated that some embodiments of the invention includethe peptides that comprise at least a fragment of a derivative of SEQ IDNO: 3 of at least 4 amino acids rather than a fragment of SEQ ID NO:3and/or a derivative of SEQ ID NO:5 rather than SEQ ID NO:5. As usedherein, the term "derivative of SEQ ID NO:3" is meant to refer to apeptide which comprises an amino acid sequence substantially identicalto SEQ ID NO:3. As used herein, the term "derivative of SEQ ID NO:5" ismeant to refer to a peptide which comprises an amino acid sequencesubstantially identical to SEQ ID NO:5. As used herein, the term"substantially identical to SEQ ID NO:3 or a fragment thereof" refers toan amino acid sequence that is the same as the amino acid sequence ofSEQ ID NO:3 or a fragment thereof except some of the residues aredeleted, substituted with conservative amino acids or additional aminoacids are inserted. As used herein, the term "substantially identical toSEQ ID NO:5" refers to an amino acid sequence that is the same as theamino acid sequence of SEQ ID NO:5 except some of the residues aredeleted, substituted with conservative amino acids or additional aminoacids are inserted. Those having ordinary skill in the art can readilydesign and produce derivatives having substantially identical amino acidsequences of SEQ ID NO:3 or a fragment thereof or SEQ ID NO:5 withdeletions and/or insertions and/or conservative substitutions of aminoacids. For example, following what are referred to as Dayhof's rules foramino acid substitution (Dayhof, M. D. (1978) Nat. Biomed. Res. Found.,Washington, D.C. Vol. 5, supp. 3), amino acid residues in a peptidesequence may be substituted with comparable amino acid residues. Suchsubstitutions are well known and are based the upon charge andstructural characteristics of each amino acid. Derivatives includefragments of SEQ ID NO:3 or SEQ ID NO:5 with deletions and/or insertionsand/or conservative substitutions.

Peptides may be synthesized by well known techniques such as thesolid-phase synthetic technique initially described by Merrifield, in J.Am. Chem. Soc., 15: 2149-2154 (1963). Other peptide synthesis techniquesmay be found, for example, in M. Bodanszky et al., (1976) PeptideSynthesis, John Wiley & Sons, 2d Ed.; Kent and Clark-Lewis in SyntheticPeptides in Biology and Medicine, p. 295-358, eds. Alitalo, K., et al.Science Publishers, (Amsterdam, 1985); as well as other reference worksknown to those skilled in the art. A summary of peptide synthesistechniques may be found in J. Stuart and J. D. Young, Solid PhasePeptide Synthelia, Pierce Chemical Company, Rockford, Ill. (1984), whichis incorporated herein by reference. The synthesis of peptides bysolution methods may also be used, as described in The Proteins, Vol.II, 3d Ed., p. 105-237, Neurath, H. et al., Eds., Academic Press, NewYork, N. Y. (1976). Appropriate protective groups for use in suchsyntheses will be found in the above texts, as well as in J. F. W.McOmie, Protective Groups in Organic Chemistry, Plenum Press, New York,N.Y. (1973), which is incorporated herein by reference.

In general, these synthetic methods involve the sequential addition ofone or more amino acid residues or suitable protected amino acidresidues to a growing peptide chain. Normally, either the amino orcarboxyl group of the first amino acid residue is protected by asuitable, selectively removable protecting group. A different,selectively removable protecting group is utilized for amino acidscontaining a reactive side group, such as lysine.

Using a solid phase synthesis as an example, the protected orderivatized amino acid is attached to an inert solid support through itsunprotected carboxyl or amino group. The protecting group of the aminoor carboxyl group is then selectively removed and the next amino acid inthe sequence having the complementary (amino or carboxyl) group suitablyprotected is admixed and reacted with the residue already attached tothe solid support. The protecting group of the amino or carboxyl groupis then removed from this newly added amino acid residue, and the nextamino acid (suitably protected) is then added, and so forth. After allthe desired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to provide the final peptide.The peptide of the invention is preferably devoid of benzylated ormethylbenzylated amino acids. Such protecting group moieties may be usedin the course of synthesis, but they are removed before the peptides areused. Additional reactions may be necessary, as described elsewhere, toform intramolecular linkages to restrain conformation.

Peptides may also be prepared by recombinant DNA techniques. Provisionof a suitable DNA sequence encoding the desired peptide permits theproduction of the peptide using recombinant techniques now known in theart. The coding sequence can be obtained from natural sources orsynthesized or otherwise constructed using widely available startingmaterials by routine methods. When the coding DNA is preparedsynthetically, advantage can be taken of known codon preferences of theintended host where the DNA is to be expressed.

To produce peptides of the invention, one having ordinary skill in theart can, using well known techniques, synthesize or obtain a DNAmolecule encoding SEQ ID NO:1 or portions thereof which include SEQ IDNO:3 or a fragment thereof or SEQ ID NO:5 from readily available humanDNA and insert that DNA molecule into a commercially availableexpression vector for use in well known expression systems. Including inthese systems are those in which the peptide of interest is produced asa fusion protein.

For example, the commercially available plasmid pSE420 (Invitrogen, SanDiego, Calif.) may be used for recombinant production in E. coli. Thecommercially available plasmid pYES2 (Invitrogen, San Diego, Calif.) maybe used for production in S. cerevisiae strains of yeast. Thecommercially available MaxBac™ (Invitrogen, San Diego, Calif.) completebaculovirus expression system may be used for production in insectcells. The commercially available plasmid pcDNA I (Invitrogen, SanDiego, Calif.) may be used for production in mammalian cells such asChinese Hamster Ovary cells.

One having ordinary skill in the art may use these or other commerciallyavailable expression vectors and systems or produce vectors using wellknown methods and readily available starting materials. Expressionsystems containing the requisite control sequences, such as promotersand polyadenylation signals, and preferably enhancers, are readilyavailable and known in the art for a variety of hosts. See e.g.,Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. ColdSpring Harbor Press (1989). Thus, the desired proteins can be preparedin both prokaryotic and eukaryotic systems, resulting in a spectrum ofprocessed forms of the protein.

The most commonly used prokaryotic system remains E. coli, althoughother systems such as B. subtilis and Pseudomonas are also useful.Suitable control sequences for prokaryotic systems include bothconstitutive and inducible promoters including the lac promoter, the trppromoter, hybrid promoters such as tac promoter, the lambda phage P1promoter. In general, foreign proteins may be produced in these hostseither as fusion or mature proteins. When the desired sequences areproduced as mature proteins, the sequence produced may be preceded by amethionine which is not necessarily efficiently removed. Accordingly,the peptides and proteins claimed herein may be preceded by anN-terminal Met when produced in bacteria. Moreover, constructs may bemade wherein the coding sequence for the peptide is preceded by anoperable signal peptide which results in the secretion of the protein.When produced in prokaryotic hosts in this matter, the signal sequenceis removed upon secretion.

A wide variety of eukaryotic hosts are also now available for productionof recombinant foreign bacteria. As in bacteria, eukaryotic hosts may betransformed with expression systems which produce the desired proteindirectly, but more commonly signal sequences are provided to effect thesecretion of the protein. Eukaryotic systems have the additionaladvantage that they are able to process introns which may occur in thegenomic sequences encoding proteins of higher organisms. Eukaryoticsystems also provide a variety of processing mechanisms which result in,for example, glycosylation, carboxy-terminal amidation, oxidation orderivatization of certain amino acid residues, conformational control,and so forth.

Commonly used eukaryotic systems include, but is not limited to, yeast,fungal cells, insect cells, mammalian cells, avian cells, and cells ofhigher plants. Suitable promoters are available which are compatible andoperable for use in each of these host types as well as are terminationsequences and enhancers, as e.g. the baculovirus polyhedron promoter. Asabove, promoters can be either constitutive or inducible. For example,in mammalian systems, the mouse metallothionein promoter can be inducedby the addition of heavy metal ions.

In some embodiments, a DNA molecule that includes a nucleotide sequencewhich encodes the peptide of the invention is synthesized using theamino acid sequence information herein and the genetic code. Thosehaving ordinary skill in the art can readily synthesize DNA moleculesthat include nucleotide sequences which encode the peptides of theinvention using codons preferred by a desired host cell. The DNAmolecule that is generated may be inserted in an expression vector whichallows for very high levels of expression, sometimes referred to asoverexpression, in a desired host.

The particulars for the construction of expression systems suitable fordesired hosts are known to those in the art. For recombinant productionof the protein, the DNA encoding it is suitably ligated into theexpression vector of choice and then used to transform the compatiblehost which is then cultured and maintained under conditions whereinexpression of the foreign gene takes place. The protein of the presentinvention thus produced is recovered from the culture, either by lysingthe cells or from the culture medium as appropriate and known to thosein the art.

One having ordinary skill in the art can, using well known techniques,isolate the protein that is produced.

In addition to peptides, the present invention contemplates compoundswhich display substantially the same surface as the peptides of theinvention. Such compounds, referred to as mimetics, are not peptides butcomprise a similar surface as the peptides and can thus interact withother molecules in a similar fashion as the peptides of the invention.By providing a similar surface involved in intermolecular interactions,mimetics perform essentially the same function by essentially the samemeans to achieve essentially the same result as the peptides of theinvention.

The present invention relates to a method of therapeutically orprophylactically treating an individual suffering from or susceptible toan immediate allergic response. Those having ordinary skill in the artcan readily identify individuals suspected of suffering from or beingsusceptible to an immediate allergic response conditions such asresponse is selected from the group consisting of: graft versus hostdisease, allergic rhinitis, food allergies, atropic dermatitis andallergic asthma. In the case of GVHD, treatment may be providedprophylactically in conjunction with transplantation procedure or inresponse to symptoms associated with GVHD. Those with ordinary skill inthe art could readily identify individuals for whom administration ofthe peptides of the invention would be beneficial to alleviate orprevent immediate allergy response conditions.

The method of therapeutically or prophylactically treating an individualsuffering from or susceptible to an immediate allergic responsecomprises administering to said individual an effective amount of apeptide according to the invention. A prophylactically effective amountis an amount which is effective to prevent or decrease the immediateallergic response in an individual susceptible to such a reactionwithout causing lethal side effects on the individual. Aprophylactically effective amount is an amount which is effective todecrease or eliminate the immediate allergic response in an individualsuffering from such a reaction without causing lethal side effects onthe individual. Those having ordinary skill in the art can readily androutinely determine the ranges of both prophylactically andtherapeutically effective amounts of the peptides of the inventionwithout undue experimentation.

It is contemplated that the present invention is useful totherapeutically or prophylactically treat individuals suspected ofsuffering from or being susceptible to reactions are mediated throughthe interaction of immunoglobulin E (IgE) with the α-chain of its highaffinity Fc receptor, FcεR1.

The present invention provides pharmaceutical compositions that comprisethe peptides of the invention and pharmaceutically acceptable carriersor diluents. The pharmaceutical composition of the present invention maybe formulated by one having ordinary skill in the art. Suitablepharmaceutical carriers are described in Remington's PharmaceuticalSciences, A. Osol, a standard reference text in this field, which isincorporated herein by reference. In carrying out methods of the presentinvention, conjugated compounds of the present invention can be usedalone or in combination with other diagnostic, therapeutic or additionalagents. Such additional agents include excipients such as coloring,stabilizing agents, osmotic agents and antibacterial agents.

For parenteral administration, the peptides of the invention can be, forexample, formulated as a solution, suspension, emulsion or lyophilizedpowder in association with a pharmaceutically acceptable parenteralvehicle. Examples of such vehicles are water, saline, Ringer's solution,dextrose solution, and 5% human serum albumin. Liposomes and nonaqueousvehicles such as fixed oils may also be used. The vehicle or lyophilizedpowder may contain additives that maintain isotonicity (e.g., sodiumchloride, mannitol) and chemical stability (e.g., buffers andpreservatives). The formulation is sterilized by commonly usedtechniques. For example, a parenteral composition suitable foradministration by injection is prepared by dissolving 1.5% by weight ofactive ingredient in 0.9% sodium chloride solution.

The pharmaceutical compositions according to the present invention maybe administered as a single dose or in multiple doses. Thepharmaceutical compositions of the present invention may be administeredeither as individual therapeutic agents or in combination with othertherapeutic agents. The treatments of the present invention may becombined with conventional therapies, which may be administeredsequentially or simultaneously.

The pharmaceutical compositions of the present invention may beadministered by any means that enables the active agent to reach thetargeted cells. These methods include, but are not limited to, oral,topical, intradermal, subcutaneous, intravenous, intramuscular andintraparenteral modes of administration. The compounds may beadministered singly or in combination with other compounds. Thecompounds of the invention are preferably administered with apharmaceutically acceptable carrier selected on the basis of theselected route of administration and standard pharmaceutical practice.

The dosage administered varies depending upon factors such as:pharmacodynamic characteristics; its mode and route of administration;age, health, and weight of the recipient; nature and extent of symptoms;kind of concurrent treatment; and frequency of treatment. Usually, thedosage of peptide can be about 1 to 3000 milligrams per 50 kilograms ofbody weight; preferably 10 to 1000 milligrams per 50 kilograms of bodyweight; more preferably 25 to 800 milligrams per 50 kilograms of bodyweight. Ordinarily 8 to 800 milligrams are administered to an individualper day in divided doses 1 to 6 times a day or in sustained release formis effective to obtain desired results.

EXAMPLES Example 1 In vitro Inhibition of Binding of IgE Fc to Fcε byBiosensor Assay

The kinetics of IgE Fc binding to soluble Fcε were determined using aBIAcore biosensor (Pharmacia Biosensor AB, Uppsala, Sweden). The FcεRIαwas immobilized via primary amine groups to a carboxymethylateddextran-coated CM5 sensor chip, at a concentration of 80 μg/ml in 10 mMsodium acetate buffer pH 4.5, using N-hydroxy-succinimide andN-ethyl-N'-(3-diethyl-aminopropyl)-carbodiimide (Pharmacia AmineCoupling Kit). Free sites were blocked with 1M ethanolamine-HCl pH 8.5.Binding of peptides to IgE was measured by the ability of peptides toprevent binding of IgE to the immobilized FcεRIα. Various concentrationsof peptide were incubated with a fixed concentration of IgE (10 nM) andallowed to equilibrate for 24 h. The peptide-IgE mixtures were injectedover the sensor surface, in 10 mM HEPES buffered saline (HBS) with 150mM NaCl, 3.4 mM EDTA and 0.005% surfactant P20 (Pharmacia), at 25° C.,using conditions under which binding is limited by mass transport ratherthan by kinetic parameters. This allowed measurement of theconcentration of free IgE, and thus the solution association constant(K_(a)) for the receptor-ligand interaction. The initial rate of IgEbinding to the immobilized receptor is presented as a function ofpeptide concentration in FIG. 4.

Example 2 In vivo Test to Identify IgE-IgE Receptor Binding Inhibitors

The transplantation of B10.D2 CD4+ T-cells along with T-cell-depletedbone marrow has been shown previously to induce a lethal GVHD responsein irradiated (850 cGy) DBA/2 mice (Korngold and Sprent (1987) J. Exp.Med. 165:1552-1564, which is incorporated herein by reference). Inaddition, in this strain combination dermal mast cells are believed tobe involved in the early stages of cutaneous GVHD development. Thetransplantation of 7×10⁶ B10.D2 CD4+ T-cells resulted in a 100%incidence of lethal GVHD in DBA/2 recipients with a median survival timeof 9 days. Transplantation of donor T-cell-depleted bone marrow aloneresulted in complete survival out to termination of the experiment at 70days. To determine the ability of an L form and a reverse D form of atest compound of the invention to inhibit IgE-IgE receptor binding andthereby inhibit GVHD, DBA/2 recipients of B10.D2 CD4+ T-cells aretreated on days 0 and 3 with 0.5 mg of either test compound i.v.Incidence of survival and median survival time are compared to anuntreated group and a group treated with a scrambled reverse D form ofthe test compound. These data indicate that the ability of a reverse Dtest compound to effect the development of GVHD and alloreactivity oftransplanted donor T-cells.

Example 3

In one embodiment of the invention, a peptide that includes SEQ ID NO:3and that is restrained by cysteines at each end is synthesized. Thepeptide and has the following sequence:

C-I-Y-Y-K-D-G-E-A-L-K-Y-C^(D), where the carboxy terminal cysteine is a"D" amino acid. This D amino acid peptide is referred to herein as Damino acid peptide #1. D amino acid peptide #1 consists of a total of 13amino acids and comprises all of SEQ ID NO:3. D amino acid peptide #1 isconformationally restricted using two terminal cysteines to form adisulfide bond which cyclicizes the peptide. The D amino acid cysteineis included to facilitate the formation and stability of the disulfidebond.

Approximately 0.5×10⁶ transfected CHO cells were incubated with amixture of 5 nM labelled myeloma IgE and D amino acid peptide #1 wastitrated at various concentrations for 1 h at room temperature.Non-specific bindings were determined by pre-incubating the cells with a100-fold excess of rat IgE. After incubation, the cells were separatedfrom the unbound iodinated IgE by 2 min. centrifugation through mixedoils. The cell pellets were then counted in a 5500 Beckman gammacounter.

Concentration of the D amino acid peptide #1 used ranged form 9.324×10⁻⁸M to 3.73×10⁻⁵ M. The IC₅₀ =5.14×10⁻⁶ M. The results are shown on FIG.1.

As shown in FIG. 2, the transplantation of 2×10⁶ B10.D2 CD4+ T cellsresulted in an 83% incidence of lethal GVHD in DBA/2 recipients with amedian survival time of 15 days. Transplantation of donorT-cell-depleted bone marrow alone resulted in complete survival out totermination of the experiment at 80 days. DBA/2 recipients of B10.D2CD4+ T cells and treated on days 0 and 3 with 0.5 mg D amino acidpeptide #1 i.v. exhibited a significantly higher incidence of survival(67%) and a significantly prolonged median survival time of greater than80 days (p=0/04), compared to the untreated group. This data indicatesthat the IgE receptor analogs of the invention can diminish thedevelopment of GVHD and alloreactivity of transplanted donor T cells.

Example 4 Surface Plasmon Resonance Experiment

The kinetics of binding of IgE Fc to soluble FcεR1α were determinedusing a BIAcore biosensor (Pharmacia Biosensor AB, Uppsala, Sweden). TheFcεR1α was immobilized via primary amine groups to a carboxymethylateddextran-coated CM5 sensor chip, at a concentration of 85 μg/ml in 10mMsodium acetate buffer pH 4.5, using N-hydroxy-succinimide andN-ethyl-N'-(3-diethyl-aminopropyl)-carbodiimide. Free sites were blockedwith 1M ethanolamine-hydrochloride pH 8.5.

The association of IgE-Fc at a concentration of 5nM was monitored for 6minutes in the presence and absence of a derivative of the D amino acidpeptide #1 at various concentrations (at 24° C.) The derivative of Damino acid peptide #1 is identical to D amino acid #1, which isdescribed in Example 3, except it is biotinylated. At a concentration of2.5 mM, virtually complete inhibition of IgE-Fc binding was observed.The results are shown in FIG. 3.

Example 5

SEQ ID NO:6 is SEQ ID NO:3 with cysteine residues at the N and Cterminals. The two cysteines form disulfide bonds which result in thecyclization of the peptide.

D amino acid peptide #1 has the same amino acid sequence as SEQ ID NO:6except that the C terminus cysteine is a D amino acid. D amino acidpeptide #1 is also referred to as cyclo(L-262).

D amino acid peptide #2 has the sequence of D amino acids from aminoterminus to carboxy terminus: Cys Tyr Lys Leu Ala Glu Gly Asp Lys TyrTyr Ile CYS.sub.(L). The C terminus cysteine is an L amino acid. Thissequence, also referred to as cyclo(rD-262), is the reverse amino acidsequence of SEQ ID NO:6.

Peptides cyclo(L-262) and cyclo(rD-262) were synthesized and tested foractivity. The sequences of the peptides are as follows, cyclo(L-262)CIYYKDGEALKYC.sub.(D) (all L amino acids except the carboxy terminalcysteine) and cyclo-(rD-262) CYKLAEGDKYYIC.sub.(L) (all D amino acidsexcept carboxy terminal cysteine).

The following scrambled peptides were used as controls: cyclo(rD-scram)CLEADYKGYKYIC.sub.(L) (all D amino acids except carboxy terminalcysteine) and cyclo(L-scram) CIYKYGKYDAELC.sub.(D) (all L amino acidsexcept the carboxy terminal cysteine).

Peptides were synthesized on an Applied Biosystems 430A fully automatedpeptide synthesizer using standard fmoc chemistry. Intramoleculardisulfides were enriched by an air oxidation refolding procedure carriedout at 100 μg/ml in 50 mM ammonium bicarbonate, pH 8.5, stirredovernight at 23° C. Peptides show greater than 95% intramoleculardisulfide bonding at the end of this procedure as monitored by Ellman'sreagent and HPLC analysis. In addition, matrix assisted laserdesorption/ionization mass spectrometry (MALDI-MS) was used to confirmthe identity and cyclized status of the peptide product. Theintramolecular disulfide cyclized product was then purified by RP-HPLC.

Example 6 In vivo Inhibition of GVDH (FIG. 5)

Bone marrow cells were obtained from the femurs and tibiae of donor miceby flushing with buffered saline solution (BSS) supplemented with 0.1bovine serum albumin (Hyclone, Logan, Utah). To prepare anti-Thy-1 (Tcell-depleted) bone-marrow (ATBM), cells were incubated with J1J mAb(diluted 1:100) and complement 1:25 for 45 minutes at 37° C., followedby four washes. CD4+ T cell-enriched donor cell populations wereprepared by initially treating pooled spleen and lymph node cells withthe anti-B cell J11d mAb (diluted 1:5) and complement (1:25) for 1 hourat 37° C. This treatment generally resulted in a population between90-95% Thy-1+cells, as measured for phenotyping by flow cytometricanalysis. These cells were further treated twice with anti-CD8 mAb(3.268, diluted 1:50) plus complement (1:25) for 45 minutes at 37° C.,initially and again for 30 minutes with a wash in between. The enrichedCD4+ T cells were phenotyped by flow cytometry analysis and wereroutinely found to be negative for CD8+ T ell subset. For GVHDinduction, DBA/2 recipient mice were irradiated 900 cGy by exposure froma Gammacell ¹³⁷ Cs source (130 cGy/min). Approximately 6 hours laterthese mice were injected intravenously with donor B10.D2 ATBM cells(2×10⁶) alone as a negative control, or a mixture of ATBM (2×10⁶)together with 5×10⁶ CD4+ T cells.

To determine the ability of cyclo(L-262) and cyclo(rD-262) peptides, andscrambled versions of these peptides, to inhibit receptor binding andthereby inhibit GVHD, DBA/2 recipients of B10.D2 CD4+ T-cells weretreated on days 0 and 3 with 0.5 mg of peptide i.v. Mice were observeddaily for mortality until 70 days post-transplantation and weights weremonitored twice weekly. Data are shown in FIG. 5.

Example 7 In vivo Inhibition of Antigen-Induced Anaphylaxis (FIG. 6)

BALB/c mice primed to bacteriophage lambda cI repressor peptide 9-29(ref. J. Exp. Med., 174, 847, 1991), with 200 μg i.p., followed by asecondary boost 3 weeks later. They were subsequently challenged with250 μg of the same antigen, and scored for level of distress (level4=severe fatal reaction) for up to 40 minutes after challenge.Cyclo(rD-262) peptide (0.5 mg) was administered at either time 0 or 15minutes before challenge, and compared with administration of ascrambled version of the peptide at time 0. The results are shown inFIG. 6.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 6    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 193 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:1:    #Cys Val Ala Leu Leu Phe Phe Ala Pro Asp    #      15    #Gln Lys Pro Lys Val Ser Leu Asn Pro Pro    #  30    #Gly Glu Asn Val Thr Leu Thr Cys Asn Gly    #                45    #Ser Ser Thr Lys Trp Phe His Asn Gly Ser    #            60    #Ser Ser Leu Asn Ile Val Met Ala Lys Phe    #        80    #Lys Cys Gln His Gln Gln Val Asn Glu Ser    #      95    #Val Phe Ser Asp Trp Leu Leu Leu Gln Ala    #  110    #Glu Gly Gln Pro Leu Phe Leu Arg Cys His    #               125    #Val Tyr Lys Val Ile Tyr Tyr Lys Asp Gly    #           140    #Tyr Glu Asn His Asn Ile Ser Ile Thr Asn    #       160    #Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp    #      175    #Glu Pro Leu Asn Ile Thr Val Ile Lys Ala    #  190    -         Pro    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 174 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:2:    #Gly Ala Leu Gly His Gly Ile Asn Leu Asn    #      15    #Thr Asp Asp Ile Asp Glu Val Arg Trp Glu    #  30    #Ala Glu Glu Lys Arg Lys Met Lys Pro Phe    #                45    #Glu Ile Leu Ala Asn Gly Asp Leu Lys Ile    #            60    #Asp Ser Gly Thr Tyr Asn Val Thr Val Tyr    #        80    #Ile Leu Asn Lys Ala Leu Asp Leu Arg Ile    #      95    #Pro Met Ile Tyr Trp Glu Cys Ser Asn Ala    #  110    #Leu Glu Gly Thr Asp Val Glu Leu Lys Leu    #               125    #Leu Arg Ser Leu Arg Gln Lys Thr Met Ser    #           140    #Arg Ala Pro Phe Lys Cys Lys Ala Val Asn    #       160    #Glu Met Glu Val Val Asn Cys Pror    #      170    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 11 amino              (B) TYPE: amino acid              (D) TOPOLOGY: both    -     (ii) MOLECULE TYPE: peptide    -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:3:    #Glu Ala Leu Lys Tyrr Lys Asp Gly    #      10    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 19 amino              (B) TYPE: amino acid              (D) TOPOLOGY: both    -     (ii) MOLECULE TYPE: peptide    -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:4:    #Lys Val Trp Gln Leu Asp Tyr Glu Ser Glu    #       15    -         Pro Leu Asn    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 6 amino              (B) TYPE: amino acid              (D) TOPOLOGY: both    -     (ii) MOLECULE TYPE: peptide    -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:5:    -        Lys Val Trp Gln Leu Asp    #    5 1    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 13 amino              (B) TYPE: amino acid              (D) TOPOLOGY: both    -     (ii) MOLECULE TYPE: peptide    -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:6:    #Gly Glu Ala Leu Lys Tyr Cyss Asp    #      10    __________________________________________________________________________

We claim:
 1. A peptide consisting of up to 25 amino acids, wherein saidpeptidea) comprises at least a fragment of SEQ ID NO:3 of at least 6amino acids and b) is capable of modulating IgE binding to FcεR1.
 2. Thepeptide of claim 1 wherein said peptide consists of 8-15 amino acids. 3.The peptide of claim 1 wherein said peptide comprises SEQ ID NO:3 or afragment thereof selected from the group consisting of amino acids 1-6,2-7, 3-8, 4-9, 5-10, 6-11, 1-7, 2-8, 3-9, 4-10, 5-11, 1-8, 2-9, 3-10,4-11, 1-9, 2-10, 3-11, 1-10, and 2-11 of SEQ ID NO:3.
 4. The peptide ofclaim 1 wherein said peptide is conformationally restricted.
 5. Thepeptide of claim 1 having the amino acid sequenceC--Y--K--L--A--E--G--D--K--Y--Y--I--C, wherein said peptide isconstructed of all D amino acids except the amino terminal cysteine. 6.The pharmaceutical composition comprising a peptide of claim 5 and apharmaceutically acceptable carrier or diluent.
 7. A peptide consistingof up to 25 amino acids, wherein said peptidea) comprises SEQ ID NO:3and b) is capable of modulating IgE binding to FcεR1.
 8. A peptideconsisting of up to 25 amino acids, wherein said peptidea) comprises atleast a fragment of SEQ ID NO:3 of at least 6 amino acids b) has anamino terminal cysteine and a carboxy terminal cysteine which are linkedby a disulfide bond and c) is capable of modulating IgE binding toFcεR1.
 9. The peptide of claim 8 having the amino acid sequence:C--I--Y--Y--K--D--G--E--A--L--K--Y--(D)--C, wherein the carboxy terminalcysteine is a "D" amino acid.
 10. A pharmaceutical compositioncomprising a peptide of claim 9 and a pharmaceutically acceptablecarrier or diluent.
 11. A pharmaceutical composition comprising:a) apeptide consisting of up to 25 amino acids, wherein said peptidei)comprises at least a fragment of SEQ ID NO:3 of at least 6 amino acidsand ii) is capable of modulating IgE binding to FcεR1; and b) apharmaceutically acceptable carrier or diluent.
 12. A peptide consistingof 6-50 amino acids, wherein said peptidea) comprises SEQ ID NO:5 and b)is capable of modulating IgE binding to FcεR1.
 13. The peptide of claim12 wherein said peptide consists of 6-20 amino acids.
 14. The peptide ofclaim 12 wherein said peptide consists of 8-15 amino acids.
 15. Thepeptide of claim 12 wherein said peptide comprises SEQ ID NO:4 or afragment thereof comprising amino acids 7-12, 7-13, 7-14, 7-15, 7-16,7-17, 7-18, 7-19, 6-12, 6-13, 6-14, 6-15, 6-16, 6-17, 6-18, 6-19, 5-12,5-13, 5-14, 5-15, 5-16, 5-17. 5-18, 5-19, 4-12, 4-13, 4-14, 4-15, 4-16,4-17, 4-18, 4-19, 3-12, 3-13, 3-14, 3-15, 3-16, 3-17, 3-18, 3-19, 2-12,2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 1-12, 1-13, 1-14, 1-15, 1-16,1-17, 1-18 or 1-19 of SEQ ID NO:4.
 16. The peptide of claim 12 whereinsaid peptide is conformationally restricted.
 17. The peptide of claim 12wherein said peptide has an amino terminal cysteine and a carboxyterminal cysteine which are linked by a disulfide bond.
 18. Apharmaceutical composition comprising:a) a peptide consisting of 6-50amino acids, wherein said peptidei) comprises SEQ ID NO:5 and ii) iscapable of modulating IgE binding to FcεR1; and b) a pharmaceuticallyacceptable carrier or diluent.
 19. A method of inhibiting IgE binding toFcεR1 on cells comprising the steps of:contacting cells that contain theFcεR1 with an amount of a peptide sufficient to inhibiting IgE bindingto FcεR1, wherein said peptidea) consists of 4-50 amino acids, b)comprises at least a fragment of SEQ ID NO:3 of at least 4 amino acids.20. A method of inhibiting IgE binding to FcεR1 on cells comprising thesteps of:contacting cells that contain the FcεR1 with an amount of apeptide sufficient to inhibit IgE binding to FcεR1, wherein saidpeptidea) consists of 6-50 amino acids, b) comprises SEQ ID NO:5.
 21. Amethod of therapeutically or prophylactically treating an individualsuspected of suffering from or being susceptible to an immediateallergic response comprising the steps of:administering to saidindividual an effective amount of a peptide which:a) consists of 4-50amino acids, b) comprises at least a fragment of SEQ ID NO:3 of at least4 amino acids, and c) inhibits IgE binding to FcεR1.
 22. The method ofclaim 21 wherein said immediate allergic response is selected from thegroup consisting of: graft versus host disease, allergic rhinitis, foodallergies, atropic dermatitis and allergic asthma.
 23. A method oftherapeutically or prophylactically treating an individual suspected ofsuffering from or being susceptible to an immediate allergic responsecomprising the steps of:administering to said individual an effectiveamount of a peptide which: a) consists of between 6 and 50 amino acids,b) comprises SEQ ID NO:5, and c) inhibits IgE binding to FcεR1.
 24. Themethod of claim 23 wherein said immediate allergic response is selectedfrom the group consisting of: graft versus host disease, allergicrhinitis, food allergies, atropic dermatitis and allergic asthma.
 25. Apeptide dimer comprising a first monomer and a second monomer whereina)said first monomeri) consists of 4-50 amino acids, ii) comprises atleast a fragment of SEQ ID NO:3 of at least 4 amino acids and b) saidsecond monomeri) consists of 6-50 amino acids, ii) comprises SEQ IDNO:5;wherein said dimer is capable of inhibiting IgE binding to FcεR1.26. A peptide consisting of amino and carboxy terminal cysteinesflanking SEQ ID NO:3 or a fragment thereof selected from the groupconsisting of amino acids 1-4, 2-5, 3-6, 4-7, 5-8, 6-9, 7-10, 8-11, 1-5,2-6, 3-7, 4-8, 5-9, 6-10, 7-11, 1-6, 2-7, 3-8, 4-9, 5-10, 6-11, 1-7,2-8, 3-9, 4-10, 5-11, 1-8, 2-9, 3-10, 4-11, 1-9, 2-10, 3-11, 1-10, and2-11 of SEQ ID NO:3.
 27. A pharmaceutically composition comprising:a) apeptide of claim 26 and b) a pharmaceutically acceptable carrier ordiluent.
 28. The peptide of claim 26 wherein said peptide consists ofamino and carboxy terminal cysteines flanking SEQ ID NO:3 or a fragmentthereof selected from the group consisting of amino acids 1-6, 2-7, 3-8,4-9, 5-10, 6-11, 1-7, 2-8, 3-9, 4-10, 5-11, 1-8, 2-9, 3-10, 4-11, 1-8,2-9, 3-10, 4-11, 1-9, 2-10, 3-11, 1-10, and 2-11 of SEQ ID NO:3.
 29. Apharmaceutically composition comprising:a) a peptide of claim 28 and b)a pharmaceutically acceptable carrier or diluent.